Compositions and Methods for Modulating Plant Parasitic Nematodes

ABSTRACT

Provided is a method for modulating soybean cyst nematode infestation of a plant comprising applying to the plant an effective amount of a composition comprising a  Chromobacterium, Burkholderia , and/or  Flavobacterium  strain to modulate the soybean cyst nematode infestation. The composition may be applied directly to the seed or in a substrate for growing the plant, such as in the soil.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation-in-part application of U.S.application Ser. No. 13/280,311, filed Oct. 24, 2011, which claims thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.61/406,569, filed Oct. 25, 2010. This application is also acontinuation-in-part of U.S. application Ser. No. 13/843,971, filed Mar.15, 2013, which is a continuation-in-part of U.S. application Ser. No.13/034,575, filed Feb. 24, 2011, which claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Application Nos. 61/308,287, filedFeb. 25, 2010, and 61/406,541, filed Oct. 25, 2010. This application isalso a continuation-in-part of International Application No.PCT/US13/30631, filed Mar. 13, 2013, which claims the benefit under 35U.S.C. §119(e) of U.S. Provisional Application Nos. 61/609,937, filedMar. 13, 2012, and 61/733,730, filed Dec. 5, 2012. All of theapplications are hereby incorporated by reference.

TECHNICAL FIELD

Disclosed herein are pesticidal Chromobacterium, Burkholderia, andFlavobacterium strains and compositions, as well as their methods of usefor controlling plant parasitic nematodes, particularly, soybean cystnematodes.

BACKGROUND

Nematodes are non-segmented, bilaterally symmetric, worm-likeinvertebrates that possess a body cavity and complete digestive systembut lack respiratory and circulatory systems. Their body wall contains amultilayer cuticle, a hypodermis with four longitudinal cords, andinternal musculature (Chitwood, D. J. (2003). Nematicides. Encyclopediaof Agrochemicals , vol 3. J. R. Plimmer. New York, John Wiley & Sons. 3:1104-1115). Their body contents are mostly occupied by digestive andreproductive systems. Nematodes may be classified as either parasitic orfree living. Parasitic nematodes may be classified by their hosts (e.g.,plant parasites). Free living nematodes may be classified according totheir feeding habits and include the following groups: (1) omnivores;(2) bacterial feeders; (3) fungal feeders and (4) predators.

Plant parasitic nematodes generally feed on underground parts of plants,such as roots, bulbs, and tubers as well as above ground parts of theplants, such as leaves and stems Annual crop losses caused byplant-parasitic nematodes have been estimated to exceed US $100 billion(Koenning, S. R., Overstreet, C. et al. (1999). Journal of Nematology31: 587-618). Examples of plant parasitic nematodes include but are notlimited to nematodes belonging to Meloidogyne spp. (e.g., root-knotnematodes); Pratylenchus spp. (e.g., lesion nematodes); Heterodera spp.(e.g., cyst nematodes); Globodera spp. (cyst nematodes); Ditylenchusspp. (e.g., stem and bulb nematodes); Tylenchulus spp. (e.g., citrusnematodes), Xiphinema spp. (e.g., dagger nematodes); Radopholus spp.(burrowing nematodes); Rotylenchulus spp. (e.g. reniform nematodes);Helicotylenchus spp. and Scutellonema spp. (e.g.spiral nematodes);Belonolaimus spp. (e.g., sting nematodes); Bursaphelenchus spp. (e.g.pine wilt nematodes); Hoplolaimus spp. (lance nematodes); Longidorusspp. (needle nematodes); Nacobbus spp. (false root-knot nematodes); andAphelenchoides spp. (foliar nematodes). The most efficient means forcontrolling nematodes is via nematicides that inhibit egg hatching,juvenile motility and/or plant infectivity. The development of chemicalcontrol for plant-parasitic nematodes is challenging because of bothenvironmental and physiological reasons: (1) most phytoparasiticnematodes live in a confined area in soil near the roots and hence,delivery of a chemical nematicide is difficult and (2) the outer surfaceof nematodes is a poor biochemical target, and is impermeable to manyorganic molecules (Chitwood, D. J. (2003). Nematicides. Encyclopedia ofAgrochemicals , vol 3. J. R. Plimmer. New York, John Wiley & Sons. 3:1104-1115). Moreover, delivery of toxic compounds by an oral route isnearly impossible because most plant parasitic nematode species ingestmaterial only after they have penetrated and infected plant roots.Therefore, nematicides have tended to be broad-spectrum toxins with highvolatility or with other chemical and physical properties promotingtheir motility in soil.

According to Sasser and Freckman (In J. A. Veech and D. W. Dickson(Eds.), Vistas on Nematology, 1987, (pp. 7-14). Society ofNematologists, Hyattesville), crop losses by nematodes range from 8 to20% on major crops around the world. Plant parasitic nematodes can causeconsiderable crop damage with annual losses estimated at $87 billionworldwide (Dong, L. Q. and Zhang, K. Q. (2006) Plant Soil 288: 31-45).Fumigants such as methyl bromide are very effective in controlling bothsoil-borne plant diseases and nematodes but due to the high mammaliantoxicity, ozone depleting effects and other residual effects, the use ofmethyl bromide has already been banned in various countries and itscomplete withdrawal from the market is planned by internationalagreement (Oka, Y., Nacar, S. et al. (2000). Phytopathology 90:710-715).Chemical alternatives such as methyl iodide, 1,3-Dichloropropene, andchloropicrin also have issues with mammalian and environmental safety.Chemical non-fumigant nematicides are being phased out and banned. Mostrecently, the US-EPA announced that aldicarb will be phased out.

Accordingly, there is currently a need for additional nematicides; inparticular, those with maximal nematode-specific toxicity. In addition,in light of the ever-increasing resistance of plant pathogens tosynthetic pesticides, and the environmental concerns associated withchemical pesticide use, there is a need for new, naturally-occurringpesticides (e.g., biopesticides) to which plant pathogens have notdeveloped resistance, and which have minimal environmental effects.

SUMMARY

The present invention provides natural microbial pesticides formodulating infestation of a plant by plant parasitic nematodes.

In a particular embodiment, the present invention provides a method formodulating soybean cyst nematode (H. glycines) infestation of a plantcomprising contacting the plant with an effective amount of acomposition comprising Flavobacterium sp. H492 (NRRL Accession No.B-50584) to modulate the soybean cyst nematode infestation.

In another embodiment, the present invention provides a method formodulating soybean cyst nematode infestation of a plant comprisingcontacting the plant with an effective amount of composition comprisingBurkholderia A396 (NRRL Accession No. B-50319) to modulate the soybeancyst nematode infestation.

In yet another embodiment, the present invention provides a method formodulating soybean cyst nematode infestation of a plant comprisingcontacting the plant with an effective amount of composition comprisingChromobacterium subtsugae sp. nov. (NRRL Accession No. B-30655) tomodulate the soybean cyst nematode infestation.

In a particular embodiment, the composition of the invention is a wholecell broth, cell fraction, filtrate, supernatant, compound, and/orextract of the bacterial strain. In another embodiment, the compositionis a formulation comprising the bacterial strain and inerts.

In certain embodiments, contacting the plant includes application of thecomposition to a seed of the plant prior to planting. In additionalembodiments, contacting the plant is accomplished by adding thecomposition to the substrate in which the plant is grown (e.g., soil)either before, at the time of, or after planting the seed (e.g., by soildrenching or irrigation).

In an embodiment of the invention, the methods are capable of reducingthe number of soybean cyst nematodes in the soil or plant, or on theplant, compared to a plant that not been contacted with the composition.Modulation can also include promotion of plant growth and/or increase inthe yield of seeds produced by the plant, compared to a plant that hasnot been contacted with the composition.

In a particular embodiment of the invention, the plant is a soybeanplant.

DETAILED DESCRIPTION

While the compositions and methods disclosed herein are susceptible tovarious modifications and alternative forms, exemplary embodiments willherein be described in detail. It should be understood, however, thatthere is no intent to limit the invention to the particular formsdisclosed, but on the contrary, the intention is to cover allmodifications, equivalents, and alternatives falling within the spiritand scope of the invention as defined by the appended claims.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimit of that range and any other stated or intervening value in thatstated range, is included therein. Smaller ranges are also included. Theupper and lower limits of these smaller ranges are also includedtherein, subject to any specifically excluded limit in the stated range.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs. Although any methods andmaterials similar or equivalent to those described herein can also beused in the practice or testing of the present invention, the preferredmethods and materials are now described.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “and” and “the” include plural references unless thecontext clearly dictates otherwise.

Definitions

As defined herein, “derived from” means directly isolated or obtainedfrom a particular source or alternatively having identifyingcharacteristics of a substance or organism isolated or obtained from aparticular source. In the event that the “source” is an organism,“derived from” means that it may be isolated or obtained from theorganism itself or medium used to culture or grow said organism.

As defined herein, “whole broth culture” or “whole cell broth” refers toa liquid culture containing both cells and media. A whole cell broth canalso be obtained from bacteria grown on a plate, by harvesting the cellsfrom the plate and suspending them in water or other liquid. The terms“whole broth culture” and “whole cell broth” are used interchangeably.

As defined herein, “supernatant” refers to the liquid remaining whencells grown in broth or harvested in another liquid from an agar plateand are removed by centrifugation, filtration, sedimentation, or othermeans well known in the art.

As defined herein, “filtrate” refers to liquid from a whole brothculture that has passed through a membrane.

As defined herein, “extract” refers to a liquid substance removed fromcells by a solvent (water, detergent, buffer, organic solvent) andseparated from the cells by centrifugation, filtration, phase partitionor other method.

As defined herein, “metabolite” refers to a compound, substance orbyproduct of fermentation of a microorganism, or supernatant, filtrate,or extract obtained from a microorganism that has pesticidal andparticularly, nematicidal activity.

As defined herein, an “isolated compound” is essentially free of othercompounds or substances, e.g., at least about 20% pure, preferably atleast about 40% pure, more preferably about 60% pure, even morepreferably about 80% pure, most preferably about 90% pure, and even mostpreferably about 95% pure, as determined by analytical methods,including but not limited to chromatographic methods and electrophoreticmethods.

As defined herein, “carrier” is an inert, organic or inorganic material,with which the active ingredient is mixed or formulated to facilitateits application to a plant, seed or other object to be treated, or tofacilitate its storage, transport and/or handling.

As defined herein, “modulate”, is used to mean to alter the amount ofpest infestation, plant growth, root extension, seed germination or rateof spread of pest infestation, rate of plant growth, rate of rootextension, and/or rate of seed germination. Modulation includesreduction of pest infestation, increase in plant growth, increase inroot extension, increase in seed germination, reduction of the rate ofspread of pest infestation, increase in the rate of plant growth,increase in the rate of root extension, and/or increase in rate of seedgermination.

As defined herein, “pest infestation” is the presence of a pest in anamount that causes a harmful effect including a disease or infection ina host population or emergence of an undesired pest in a growth system.

As defined herein, “pesticide” is a substance derived from a biologicalproduct or chemical substance that increase mortality or inhibit thegrowth rate of plant pests and includes but is not limited tonematicides, algaecides, herbicides, insecticides, plant fungicides,plant bactericides, and plant viricides.

As defined herein, a “plant parasitic nematode” is a nematode that feedson and causes injury on any part of a plant.

As defined herein, “a non-parasitic nematode” is a nematode that existsindependently in the soil and does not cause damage or injury to anypart of a plant.

As defined herein, “substrate” is a surface or medium in which a plantgrows. Substrate includes, but is not limited to, soil, an artificialgrowth surface or medium, water, and sediment.

Chromobacterium

A strain of Chromobacterium sp., particularly a strain ofChromobacterium subtsugae and more particularly, a strain ofChromobacterium subtsugae sp. nov. and even more particularly a strainof Chromobacterium subtsugae sp. nov. having the identifyingcharacteristics of NRRL Accession No. B-30655, is described in U.S. Pat.No. 7,244,607. Methods of growing the Chromobacterium sp. and making acomposition of Chromobacterium sp. and its products (e.g., a whole-cellbroth) are described, for example, in U.S. Pub. No. 2012-0100236.

Burkholderia

A non-Burkholderia cepacia complex, non-Burkholderia plantari,non-Burkholderia gladioli, Burkholderia sp., in particular, BurkholderiaA396 sp. nov. rinojensis (NRRL Accession No. B-50319), that isnon-pathogenic to vertebrates, such as birds, mammals and fish, isdescribed in, for example, U.S. Pub. No. 2011-0207604 and U.S. App. No.13/843,971. Methods of growing the Burkholderia sp. and making acomposition of Burkholderia sp. and its products (e.g., a whole-cellbroth) are also described in U.S. Pub. No. 2011-0207604 and U.S.application Ser. No. 13/843,971.

Flavobacterium

Flavobacterium sp., a gram negative bacterium, is a member of theFlavobacteriaceae family. In particular, the Flavobacterium species isFlavobacterium sp. H492 (NRRL Accession No. B-50584), as described inPCT App. No. PCT/U.S. Ser. No. 13/30631. Methods of growing theFlavobacterium sp. and making a composition of the Flavobacterium sp.and its products (e.g., a whole-cell broth) are also described in PCTApp. No. PCT/U.S. Ser. No. 13/30631.

Compositions

Compositions comprise a strain from a Chromobacterium sp., Burkholderiasp., and/or Flavobacterium sp. The compositions may be a substantiallypure culture, whole broth culture, liquid culture, suspension, cellfraction, supernatant, filtrate, extract, or compound of the bacterialstrain, or combinations of the foregoing which in particular havenematicidal activity.

The compositions set forth above can be formulated in any manner.Non-limiting formulation examples include, but are not limited to,emulsifiable concentrates (EC), wettable powders (WP), soluble liquids(SL), aerosols, ultra-low volume concentrate solutions (ULV), solublepowders (SP), microencapsulation, water dispersed granules, flowables(FL), microemulsions (ME), nano-emulsions (NE), seed treatments, etc. Inany formulation described herein, percent of the active ingredient iswithin a range of about 0.01% to 99.99%.

The compositions may be in the form of a liquid, gel or solid. A solidcomposition can be prepared by suspending a solid carrier in a solutionof active ingredient(s) and drying the suspension under mild conditions,such as evaporation at room temperature or vacuum evaporation at 65° C.or lower. A composition may comprise gel-encapsulated activeingredient(s). Such gel-encapsulated materials can be prepared by mixinga gel-forming agent (e.g., gelatin, cellulose, or lignin) with a cultureor suspension of live or inactivated bacterial strain of the presentinvention, or a cell-free filtrate or cell fraction of the bacterialculture or suspension, or a spray- or freeze-dried culture, cell, orcell fraction or in a solution of pesticidal compounds used in themethod of the invention; and inducing gel formation of the agent.

The composition may additionally comprise a surfactant to be used forthe purpose of emulsification, dispersion, wetting, spreading,integration, disintegration control, stabilization of activeingredients, and improvement of fluidity or rust inhibition. In aparticular embodiment, the surfactant is a non-phytotoxic non-ionicsurfactant which preferably belongs to EPA List 4B. In anotherparticular embodiment, the nonionic surfactant is polyoxyethylene (20)sorbitan monolaurate. The concentration of surfactants may range betweenabout 0.1-35% of the total formulation; a preferred range is about5-25%. The choice of dispersing and emulsifying agents, such asnon-ionic, anionic, amphoteric and cationic dispersing and emulsifyingagents, and the amount employed is determined by the nature of thecomposition and the ability of the agent to facilitate the dispersion ofthe compositions of the present invention.

The composition set forth above may contain one or more of aChromobacterium sp., Burkholderia sp., or Flavobacterium sp. and may becombined with another microorganism and/or pesticide (e.g., nematicide,fungicide, insecticide, herbicide). The microorganism may include, butis not limited to, an agent derived from Bacillus sp. (e.g., B. firmus,B. thuringiensis, B. pumilus, B. licheniformis, B. amyloliquefaciens, B.subtilis), Paecilomyces sp. (P. lilacinus), Pasteuria sp. (P.penetrans), Pseudomonas sp., Brevabacillus sp., Lecanicillium sp.,Ampelomyces sp., Pseudozyma sp., Streptomyces sp. (S. bikiniensis, S.costaricanus, S. avermitilis), Trichoderma sp., Gliocladium sp.,avermectin, Myrothecium sp., Paecilomyces spp., Sphingobacterium sp.,Arthrobotrys sp., Chlorosplrnium sp, Neobulgaria sp, Daldinia sp,Aspergillus sp, Chaetomium sp, Lysobacter sp, Lachnum papyraceum,Verticillium suchlasporium, Arthrobotrys oligospora, Pochoniachlamydosporia (synonym: Verticillium chlamydosporium), Hirsutellaminnesotensis, Hirsutella rhossiliensis, Pleurotus ostreatus, Omphalotusolearius, Lampteromyces japonicas, Brevudimonas sp., and Muscodor sp.

The pesticide may be a natural oil, oil product or chemical pesticide.In particular, the agent may be a natural oil or oil-product havingnematicidal, fungicidal and/or insecticidal activity (e.g., paraffinicoil, tea tree oil, lemongrass oil, clove oil, cinnamon oil, citrus oil(including but not limited to bitter orange, orange, lemon) rosemaryoil, pyrethrum, allspice, bergamot, blue gum, camomile, citronella,common jasmine, common juniper, common lavender, common myrrh, fieldmint, freesia, gray santolina, herb hyssop, holy basil, incense tree,jasmine, lavender, marigold, mint, peppermint, pot marigold, spearmint,ylang-ylang tree, and saponins.

The chemical pesticide may be a single site anti-fungal agent which mayinclude but is not limited to benzimidazole, a demethylation inhibitor(DMI) (e.g., imidazole, piperazine, pyrimidine, triazole), morpholine,hydroxypyrimidine, anilinopyrimidine, phosphorothiolate, quinone outsideinhibitor, quinoline, dicarboximide, carboximide, phenylamide,anilinopyrimidine, phenylpyrrole, aromatic hydrocarbon, cinnamic acid,hydroxyanilide, antibiotic, polyoxin, acylamine, phthalimide, benzenoid(xylylalanine), a demethylation inhibitor selected from the groupconsisting of imidazole, piperazine, pyrimidine and triazole (e.g.,bitertanol, myclobutanil, penconazole, propiconazole, triadimefon,bromuconazole, cyproconazole, diniconazole, fenbuconazole, hexaconazole,tebuconazole, tetraconazole), myclobutanil, and a quinone outsideinhibitor (e.g., strobilurin). The strobilurin may include but is notlimited to azoxystrobin, kresoxim-methoyl or trifloxystrobin. In yetanother particular embodiment, the anti-fungal agent is a quinone, e.g.,quinoxyfen (5,7-dichloro-4-quinolyl 4-fluorophenyl ether). Theanti-fungal agent may also be derived from a Reynoutria extract. Thechemical pesticide can also be a multi-site non-inorganic, chemicalfungicide. For example, the chemical fungicide may be chloronitrile,quinoxaline, sulphamide, phosphonate, phosphite, dithiocarbamate,chloralkythios, phenylpyridin-amine, or cyano-acetamide oxime.

Nematicides may include, but are not limited to, avermectin nematicides(e.g., abamectin); botanical nematicides (e.g., carvacrol); carbamatenematicides (e.g., benomyl carbofuran, carbosulfan, cloethocarb); oximecarbamate nematicides (e.g., alanycarb, aldicarb aldoxycarb, oxamyltirpate); fumigant nematicides (e.g., carbon disulfide, cyanogen,1,2-dichloropropane, 1,3-dichloropropene, dithioether, methyl bromide,methyl iodide, sodium tetrathiocarbonate); organophosphorus nematicides,which includes, but are not limited to, organophosphate nematicides(e.g., diamidafos, fenamiphos, fosthietan, phosphamidon);organothiophosphate nematicides (e.g., cadusafos, chlorpyrifos,dichlofenthion dimethoate ethoprophos, fensulfothion, fosthiazate,heterophos, isamidofos, isazofos, phorate, phosphocarb, terbufos,thionazin, triazophos); phosphonothioate nematicides (e.g., imicyafos,mecarphon); and other nematicides (e.g., acetoprole, benclothiaz,chloropicrin, dazomet, DBCP, DCIP, fluensulfone, furfural, metam, methylisothiocyanate, xylenols, spirotetramat).

The compositions disclosed herein can also be used in combination withother growth promoting agents such as synthetic or organic fertilizers(e.g., di-ammonium phosphate in either granular or liquid form), compostteas, seaweed extracts, plant growth hormones such as IAA (indole aceticacid) used in a rooting hormone treatment for transplants either aloneor in combination with plant growth regulators such as IBA (indolebutyric acid) and NAA (naphthalene acetic acid), and growth promotingmicrobes, such as Bacillus spp., Pseudomonads, Rhizobia, and Trichodermaspp.

Furthermore, the compositions can be used in combination withseed-coating agents. Such seed coating agents include, but are notlimited to, ethylene glycol, carboxymethyl cellulose, methyl cellulose,polyethylene glycol, chitosan, carboxymethyl chitosan, peat moss, resinsand waxes. The compositions may be applied using methods known in theart. Specifically, these compositions may be applied to and aroundplants or plant parts. Plants are to be understood as meaning in thepresent context all plants and plant populations such as desired andundesired wild plants or crop plants (including naturally occurring cropplants). Crop plants can be plants which can be obtained by conventionalplant breeding and optimization methods or by biotechnological andgenetic engineering methods or by combinations of these methods,including the transgenic plants and including the plant cultivarsprotectable or not protectable by plant breeders' rights. Plants includeall parts and organs of plants above and below the ground, such asshoot, leaf, flower and root, examples which may be mentioned beingleaves, needles, stalks, stems, flowers, fruit bodies, fruits, seeds,roots, tubers and rhizomes. The plants include, but are not limited to,harvested material, and vegetative and generative propagation material,for example cuttings, tubers, rhizomes, offshoots and seeds. Plants thatmay be treated include but are not limited to: (A) major edible foodcrops, which include but are not limited to (1) cereals (e.g., Africanrice, barley, durum wheat, einkorn wheat, emmer wheat, finger millet,foxtail millet, hairy crabgrass, Indian barnyard millet, Japanesebarnyard millet, maize, nance, oat, pearl millet, proso millet, rice,rye, sorghum, Sorghum spp., rye, spelt wheat); (2) fruits (e.g., abiu,acerola, achacha, African mangosteen, alpine currant, ambarella,American gooseberry, American persimmon, apple, apricot, arazá, Asianpalmyra palm, Asian pear, atemoya, Australian desert raisin, avocado,azarole, babaco, bael, banana, Barbados gooseberry, bergamot, betel nut,bignay, bilberry, bilimbi, binjai, biriba, bitter orange, blackchokeberry, black mulberry, black sapote, blackberry, blue-berriedhoneysuckle, borojó, breadfruit, murmese grape, button mangosteen,cacao, calamondin, canistel, cantaloupe, cape gooseberry, cashew nut,cassabanana, cempedak, charichuelo, cherimoya, cherry, cherry of the RioGrande, cherry plum, Chinese hawthorn, Chinese white pear, chokeberry,citron, cocona, coconut, cocoplum, coffee, coffee Arabica, coffeerobusta, Costa Rica pitahaya, currants, custard apple, date, date-plum,dog rose, dragonfruit, durian, elderberry, elephant apple, Ethiopianeggplant, European nettle tree, European wild apple, feijoa, fig, gac,genipapo, giant granadilla, gooseberry, goumi, grape, grapefruit, greatmorinda, greengage, guava, hardy kiwi, hog plum, horned melon, horsemango, Indian fig, Indian jujube, jabuticaba, jackberry, jackfruit,Japanese persimmon, Japanese wineberry, jocote, jujube, kaffir lime,karanda, kei apple, kepel apple, key lime, kitembilla, kiwi fruit,korlan, kubal vine, kuwini mango, kwai muk, langsat, large cranberry,lemon, Liberian coffee, longan, loquat, lychee, malay apple, mameysapote, mammee apple, mango, mangosteen, maprang, marang, medlar, melon,Mirabelle plum, miracle fruit, monkey jack, moriche palm, mountainpapaya, mountain soursop, mulberry, naranjilla, natal plum, northernhighbush blueberry, olive, otaheite gooseberry, oval kumquat, papaya,para guava, passionfruit, pawpaw, peach, peach-palm, pear, pepino,pineapple, pitomba Eugenia luschnathiana, pitomba talisia esculenta,plantain, plum, pomegranate, pomelo, pulasan, purple chokeberry, quince,rambutan, ramontchi, raspberry, red chokeberry, red currant, redmulberry, red-fruited strawberry guava, rhubarb, rose apple, roselle,safou, salak, salmonberry, santol, sapodilla, satsuma, seagrape,soncoya,sour cherry, soursop, Spanish lime, Spanish tamarind, starapple, starfruit, strawberry, strawberry guava, strawberry tree, sugarapple, Surinam cherry, sweet briar, sweet granadilla, sweet lime,tamarillo, tamarind, tangerine, tomatillo, tucuma palm, Vaccinium spp.,velvet apple, wampee, watermelon, watery rose apple, wax apple, whitecurrant, white mulberry, white sapote, white star apple, wolfberry(Lyceum barbarum, L. chinense), yellow mombin, yellow pitaya,yellow-fruited strawberry, guava); (3) vegetables (e.g., ackee, agate,air potato, Amaranthus spp., American groundnut, antroewa, armeniancucumber, arracacha, arrowleaf elephant ear, arrowroot, artichoke, ashgourd, asparagus, avocado, azuki bean, bambara groundnut, bamboo,banana, barbados gooseberry, beet, beet root, bitter gourd, bittervetch, bitterleaf, black mustard, black radish, black salsify, blanchedcelery, breadfruit, broad bean, broccoli, brussels sprout, Buck's hornplantain, buttercup squash, butternut squash, cabbage, caigua, calabash,caraway seeds, carob, carrot, cassabanana, cassava, catjang,cauliflower, celeriac, celery, celtuce, chard, chayote, chickpea,chicory, chilacayote, chili pepper (Capsicum annuum, C. baccatum, C.chinense, C. frutescens, C. pubescens), Chinese cabbage, Chinese waterchestnut, Chinese yam, chives, chufa sedge, cole crops, common bean,common purslane, corn salad, cowpea, cress, cucumber, cushaw pumpkin,drumstick tree, eddoe, eggplant, elephant foot yam, elephant garlic,endive, enset, Ethiopian eggplant, Florence fennel, fluted gourd, gac,garden rocket, garlic, geocarpa groundnut, good king henry, grass pea,groundut, guar bean, horse gram, horseradish, hyacinth bean, iceplant,Indian fig, Indian spinach, ivy gourd, Jerusalem artichoke, jicama,jute, kale, kohlrabi, konjac, kurrat, leek, lentil, lettuce, Lima bean,lotus, luffa, maca, maize, mangel-wurzel, mashua, moso bamboo, mothbean, mung bean, napa cabbage, neem, oca, okra, oldham's bamboo, olive,onion, parsnip, pea, pigeon pea, plantain, pointed gourd, potato,pumpkins, squashes, quinoa, radish, rapeseed, red amaranth, rhubarb,ribbed gourd, rice bean, root parsley, runner bean, rutabaga, sago palm,salsify, scallion, sea kale, shallot, snake gourd, snow pea, sorrel,soybean, spilanthes, spinach, spinach beet, sweet potato, taro, tarwi,teasle gourd, tepary bean, tinda, tomato, tuberous pea, turnip,turnip-rooted chervil, urad bean, water caltrop trapa bicornis, watercaltrop trapa natans, water morning slory, watercress, welsh onion, westAfrican okra, west Indian gherkin, white goosefoot, white yam, wingedbean, winter purslane, yacón, yam, yard-long bean, zucchinietables); (4)Food crops (e.g., abiu, acerola, achacha, ackee, African mangosteen,African rice, agate, air potato, alpine currant, Amaranthus app.,Ambarrella, American gooseberry, American groundnut, American persimmon,antroewa, apple, apricot, arazá, Armenian cucumber, arracacha, arrowleafelephant ear, arrowroot, artichoke, ash gourd, Asian palmyra palm, Asianpear, asparagus, atemoya, Australian desert raisin, avocado, azarole,azuki bean, babaco, bael, bambara groundnut, bamboo, banana, barbadosgooseberry, barley, beet, beetroot, bergamot, betel nut, bignay,bilberry, bilimbi, binjai, biriba, bitter gourd, bitter orange, bittervetch, bitterleaf, black chokeberry, black currant, black mulberry,black mustard, black radish, black salsify, black sapote, blackberry,blanched celery, blue-berried honeysuckle, borojó, breadfruit, broadbean, broccoli, Brussels sprout, Buck's horn plantain, buckwheat,Burmese grape, buttercup squash, butternut squash, button mangosteen,cabbage, cacao, caigua, calabash, calamondin, canistel, cantaloupe, capegooseberry, caraway seeds, carob, carrot, cashew nut, cassava, catjang,cauliflower, celeriac, celery, celtuce, cempedak, chard, charichuelo,chayote, cherimoya, cherry, cherry of the Rio Grande, cherry plum,chickpea, chicory, chilacayote, chili pepper (Capsicum annuum, C.baccatum, C. chinense, C. frutescens, C. pubescens), Chinese cabbage,Chinese hawthorn, Chinese water chestnut, Chinese white pear, Chineseyam, chives, chokeberry, chufa sedge, citron, cocona, coconut, cocoplum,coffee, coffee (Arabica and Robusta types), cole crops, common bean,common purslane, corn salad, Costa Rica pitahaya, cowpea, cress,cucumber, currants, cushaw pumpkin, custard apple, date, date-plum, dogrose, dragonfruit, drumstick tree, durian, durum wheat, eddoe, eggplant,einkorn wheat, elderberry, elephant apple, elephant foot yam, elephantgarlic, emmer wheat, endive, enset, Ethiopian eggplant, European nettletree, European wild apple, feijoa, fig, finger millet, florence fennel,fluted gourd, foxtail millet, gac, garden rocket, garlic, genipapo,geocarpa groundut, giant granadilla, good king henry, gooseberry, goumi,grape, grapefruit, grass pea, great morinda, greengage, groundnut,grumichama, guar bean, guava, hairy crabgrass, hardy kiwi, hog plum,horned melon, horse gram, horse mango, horseradish, hyacinth bean,iceplant, Indian barnyard millet, Indian fig, Indian jujube, Indianspinach, ivy gourd, jabuticaba, jackalberry, jackfruit, jambul, Japanesebarnyard millet, Japanese persimmon, Japanese wineberry, Jerusalemartichoke, jocote, jujube, jute, kaffir lime, kale, karanda, kei apple,kepel apple, key lime, kitembilla, kiwifruit, kohlrabi, konjac, korlan,kubal vine, kurrat, kuwini mango, kwai muk, langsat, large cranberry,leek, lemon, lentil, lettuce, Liberian coffee, lima bean, longan,loquat, lotus, luffa, lychee, maca, maize, malay apple, mamey saptoe,mammee apple, mangel-wurzel, mango, mangosteen, maprang, marang, mashua,medlar, melon, Mirabelle plum, miracle fruit, monk fruit, monkey jack,moriche palm, moso bamboo, moth bean, mountain papaya, mountain soursop,mulberry, mung bean, mushrooms, nance, napa cabbage, naranjilla, natalplum, neem, northern highbush blueberry, oat, oca, oil palm, okra,oldman's bamboo, olive, onion, orange, otaheite gooseberry, ovalkumquat, papaya, para guava, parsnip, passionfruit, pawpaw, pea, peach,peach-palm, pear, pearl millet, pepino, pigeon pea, pineapple, Pitomba(Eugenia luschnathiana, Talisia esculenta), plantain, plum, pointedgourd, pomegranate, pomelo, potato, proso millet, pulasan, pumpkins andsquashes, purple chokeberry, quince, quinoa, radish, rambutan,ramontchi, rapeseed, raspberry, red amaranth, red chokeberry, redcurrant, red mulberry, red-fruited strawberry guava, rhubarb, ribbedgourd, rice, rice bean, root parsley, rose apple, roselle, runner bean,rutabaga, rye, safou, sago palm, salak, salmonberry, salsify, santol,sapodilla, Satsuma, scallion, sea kale, seagrape, shallot, snake gourd,snow pea, soncoya, sorghum, Sorghum spp., sorrel, sour cherry, soursop,soybean, Spanish lime, Spanish tamarind, spelt wheat, spilanthes,spinach, spinach beet, star apple, starfruit, strawberry, strawberryguava, strawberry tree, sugar apple, sugar beet, sugarcane, surinamcherry, sweet briar, sweet granadilla, sweet lime, sweet potato,tamarillo, tamarind, tangerine, taro, tarwi, teasle gourd, tef, teparybean, tinda, tomatillo, tomato, tuberous pea, tucuma palm, turnip,turnip-rooted chervil, urad bean, Vaccinium spp., velvet apple, wampee,water caltrop (Trapa bicornis, T. natans), water morning glory,watercress, watermelon, watery rose apple, wax apple, welsh onion, westAfrican okra, west Indian gherkin, wheat, white currant, whitegoosefoot, white mulberry, white sapote, white star apple, white yam,winged bean, winter purslane, wolfberry (Lycium barbarum, L. chinense),yacon, yam, yangmei, yard-long bean, yellow mombin, yellow pitaya,yellow-fruited strawberry guava, zucchini; (B) Other edible crops, whichincludes but is not limited to (1) Herbs (e.g., Absinthium, alexanders,basil, bay laurel, betel nut, camomile, chervil, chili pepper (Capsicumannuum, C. baccatum, C. chinense, C. frutescens, C. pubescens), chilipeppers, chives, cicely, common rue, common thyme, coriander, cress,culantro, curly leaf parsley, dill, epazote, fennel, flat leaf parsley,ginseng, gray santolina, herb hyssop, holy basil, hop, jasmine, kaffirlime, lavender, lemon balm, lemon basil, lemon grass, lovage, marjoram,mint, oregano, parsley, peppermint, perilla, pot marigold, rooibos,rosemary, sage, shiny-leaft buckthorn, sorrel, spearmint, summer savory,tarragon, Thai basil, valerian, watercress, wild betel, winter savory,yerba mate); (2) Spices (e.g., ajowan, allspice, anise, bay laurel,black cardamom, black mustard, black pepper, caper, caraway seeds,cardamom, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C.frutescens, C. pubescens), chili peppers, cinnamon, clove, commonjuniper, coriander, cumin, fennel, fenugreek, garlic, ginger, kaffirlime, liquorice, nutmeg, oregano, pandan, parsley, saffron, star anise,turmeric, vanilla, white mustard); (3) Medicinal plants (e.g.,absinthium, alfalfa, aloe vera, anise, artichoke, basil, bay laurel,betel leat, betel nut, bilberry, black cardamom, black mustard, blackpepper, blue gum, borojó, camomlie, caper, cardamom, castor bean, chilipeppers, Chinese yam, chives, cola nut, common jasmine, common lavender,common myrrh, common rue, cilantro, cumin, dill, dog rose, epazote,fennel, fenugreek, gac, garlic, ginger, gray santolina, gum Arabic, herbhyssop, holy basil, horseradish, incense tree, lavender, lemon grass,liquorice, lovage, marijuana, marjoram, monk fruit, neem, opium,oregano, peppermint, pot marigold, quinine, red acacia, red currant,rooibos, safflower, sage, shiny-leaf buckthorn, sorrel, spilanthes, staranise, tarragon, tea, turmeric, valerian, velvet bean, watercress, whitemustard, white sapote, wild betel, wolfberry (Lycium barbarum, L.chinense), yerba mate); (4) Stimulants (e.g., betel leaf, betel nut,cacao, chili pepper (Capsicum annuum, C. baccatum, C. chinense, C.frutescens, C. pubescens), chili peppers, coffee, coffee (Arabica,Robusta), cola nut, khat, Liberian coffee, tea, tobacco, wild betel,yerba mate); (5) Nuts (e.g., almond, betel nut, Brazil nut, cashew nut,chestnut, Chinese water chestnut, coconut, cola nut, common walnut,groundnut, hazelnut, Japanese stone oak, macadamia, nutmeg, paradisenut, pecan nut, pistachio nut, walnut); (6) Edible seeds (e.g., blackpepper, Brazil nut, chilacayote, cola nut, fluted gourd, lotus, opium,quinoa, sesame, sunflower, water caltrop (Trapa bicornis, T. natans);(7) Vegetable oils (e.g., black mustard, camelina, castor bean, coconut,cotton, linseed, maize, neem, niger seed, oil palm, olive, opium,rapeseed, safflower, sesame, soybean, sunflower, tung tree, turnip); (8)Sugar crops (e.g., Asian palmyra palm, silver date palm, sorghum, sugarbeet, sugarcane); (9) Pseudocereals (e.g., Amaranthus spp., buckwheat,quinoa, red amaranth); (10) Aphrodisiacs (e.g., borojó, celery, durian,garden rocket, ginseng, maca, red acacia, velvet bean); (C) Non foodcategories, including but not limited to (1) forage and dodder crops(e.g., agate, alfalfa, beet, broad bean, camelina, catjang, grass pea,guar bean, horse gram, Indian barnyard millet, Japanese barnyard millet,lespedeza, lupine, maize, mangel-wurzel, mulberry, niger seed, rapeseed,rice bean, rye); (2) Fiber crops (e.g., coconut, cotton, fique, hemp,henequen, jute, kapok, kenaf, linseed, manila hemp, New Zealand flax,ramie, roselle, sisal, white mulberry); (3) Energy crops (e.g., bluegum, camelina, cassava, maize, rapeseed, sorghum, soybean, Sudan grass,sugar beet, sugarcane, wheat); (4) Alcohol production, (e.g., barley,plum, potato, sugarcane, wheat, sorghum); (5) Dye crops (e.g., chayroot, henna, indigo, old fustic, safflower, saffron, turmeric); (6)Essential oils (e.g., allspice, bergamot, bitter orange, blue gum,camomile, citronella, clove, common jasmine, common juniper, commonlavender, common myrrh, field mint, freesia, gray santolina, herbhyssop, holy basil, incense tree, jasmine, lavender, lemon, marigold,mint, orange, peppermint, pot marigold, spearmint, ylang-ylang tree);(6) Green manures (e.g., alfalfa, clover, lacy Phacelia, sunn hemp,trefoil, velvet bean, vetch); (7) Erosion prevention (e.g., bamboo,cocoplum); (8) Soil improvement (e.g., lupine, vetch); (9) Cover crops(e.g., Alfalfa, lacy Phacelia, radish); (10) Botanical pesticides (e.g.,jicama, marigold, neem, pyrethrum); (11) Cut flowers (e.g., carnation,chrysanthemum, daffodil, dahlia, freesia, gerbera, marigold, rose,sunflower, tulip); (12) Ornamental plants (e.g., African mangosteen,aloe vera, alpine currant, aster, black chokeberry, breadfruit,calamondin, carnation, cassabanana, castor bean, cherry plum,chokeberry, chrysanthemum, cocoplum, common lavender, crocus, daffodil,dahlia, freesia, gerbera, hyacinth, Japanese stone oak, Jasmine, lacyPhacelia, lotus, lupine, marigold, New Zealand flax, opium, purplechokeberry, ramie, red chokeberry, rose, sunflower, tulip, whitemulberry); (D) Trees which include but are not limited to abelia,almond, apple, apricot, arborvitae nigra american, arborvitae, ash,aspen, azalea, baldcypress, beautybush, beech, birch, black tupelo,blackberry, blueberry, boxwood, buckeye, butterfly bush, butternut,camellia, catalpa, cedar, cherry, chestnut, coffeetree, crab trees,crabapple, crapemyrtle, cypress, dogwood, douglasfir, ebony, elderAmerican, elm, fir, forsythia, ginkgo, goldenraintree, hackberry,hawthorn, hazelnut, hemlock, hickory, holly, honeylocust, horsechestnut,hydrangea, juniper, lilac, linden, magnolia, maple, mockorange,mountainash, oak, olive, peach, pear, pecan, pine, pistache, planetree,plum, poplar, pivet, raspberry, redbud, redcedar, redwood, rhododendron,rose-of-sharon, sassafras, sequoia, serviceberry, smoketree, soapberry,sourwood, spruce, strawberry tree, sweetshrub, sycamore, tuliptree,viburnum, walnut, weigela, willow, winterberry, witchhazel, zelkova; and(E) Turf, which includes, but is not limited to, Kentucky bluegrass,tall fescue, Bermuda grass, zoysia grass, perennial ryegrass, and finefescues (e.g. creeping red, chewings, hard, or sheep fescue).

Methods of Production

Chromobacterium sp., Burkholderia sp., and/or Flavobacterium sp. may becultivated in nutrient medium using methods known in the art. Theorganisms may be cultivated by shake flask cultivation, small scale orlarge scale fermentation (including but not limited to continuous,batch, fed-batch, or solid state fermentations) in laboratory orindustrial fermentors performed in suitable medium and under conditionsallowing cell growth. The cultivation may take place in suitablenutrient medium comprising carbon and nitrogen sources and inorganicsalts, using procedures known in the art. Suitable media may beavailable from commercial sources or prepared according to publishedcompositions.

After cultivation, a substantially pure culture or whole cell brothcomprising said strain, or cell fraction, supernatant, filtrate, orcompound (e.g., metabolite and/or extract) of or derived from saidChromobacterium sp., Burkholderia sp., and/or Flavobacterium sp. may beused in formulating a pesticidal composition. Alternatively, aftercultivation, the compounds and/or metabolites may be extracted from theculture broth.

The extract may be fractionated by chromatography. Chromatographicfractions may be assayed for toxic activity against, for example, plantparasitic nematodes, such as Heterodera glycines (soybean cystnematode), using methods known in the art. This process may be repeatedone or more times using the same or different chromatographic methods.

Uses

The compositions, cultures, supernatants, metabolites and pesticidalcompounds set forth above may be used as pesticides and in particular,may be used as nematicides, alone or in combination with one or morepesticidal substances set forth above and applied to plants, substratesfor growing plants, or seeds as set forth herein.

Specifically, nematodes that may be controlled using the method setforth above include, but are not limited to, plant parasitic nematodessuch as root-knot, reniform, cyst, and lesion nematodes, including butnot limited to Meloidogyne sp., Tylenchorhynchus sp., Belonolaimus sp.,Hoplolaimus sp., Helicotylenchus sp., Pratylenchus sp., Rotylenchulussp., Heterodera sp. (e.g., Heterodera glycines), Globodera sp.,Trichodorus sp., Paratrichodorus sp., Xiphinema sp., and Criconema sp.In particular, the plant parasitic nematodes may include but are notlimited to seed gall nematodes (Afrina wevelli), bentgrass nematodes(Anguina agrostis), shoot gall nematodes (Anguina spp.), seed gallnematodes (Anguina spp., A. amsinckiae, A. balsamophila; A. tritici),fescue leaf gall nematodes (A. graminis), ear-cockle (or wheat gall)nematodes (Anguina tritici), bud and leaf (or foliar) nematodes(Aphelenchoides spp., A. subtenuis), begonia leaf (or fern, springcrimp, strawberry foliar, strawberry nematodes, summer dwarf) nematodes(A. fragariae), fern nematodes (A. olesistus), rice nematodes (A.oryzae), currant nematodes (A. ribes), black currant (or chrysanthemum)nematodes (A. ritzemabosi), chrysanthemum foliar or leaf nematodes (A.ritzemabosi), rice white-tip (or spring dwarf, or strawberry bud)nematodes (A. besseyi), fungus-feeding (mushroom) nematodes(Aphelenchoides composticola), Atalodera spp. (Atalodera lonicerae,Atalodera ucri), spine nematodes (Bakernema variabile), sting nematodes(Belonolaimus spp., B. gracilis, B. longicaudatus), pine wood nematodes(Bursaphalenchus spp., B. xylophilus, B. mucronatus), sessile nematodes(Cacopaurus spp., C. epacris, C.pestis), amaranth cyst nematodes(Cactodera amaranthi), birch cyst nematodes (C. betulae), cactus cystnematodes (C. cacti), estonian cyst nematodes (C. estonica), Thorne'scyst nematodes (C. thornei), knotweed cyst nematodes (C. weissi), ringnematodes (Criconema spp.), spine nematodes (Criconema spp., C.civellae, C. decalineatum, C.spinalineatum), ring nematodes(Criconemella axeste, C. curvata, C. macrodora, C. parva), ringnematodes (Criconemoides spp., C. citri, C. simile), spine nematodes(Crossonema fimbriatum), eucalypt cystoid nematodes (Cryphoderaeucalypti), bud, stem and bulb nematodes (Ditylenchus spp., D. angustus,D. dipsaci, D. destructor, D. intermedius), Mushroom spawn nematodes (D.myceliophagus), awl nematodes (Dolichodorus spp., D. heterocephalus, D.heterocephalous), spear nematodes (Dorylaimus spp.), stunt nematodes(Geocenamus superbus), cyst nematodes (Globodera spp.), yarrow cystnematodes (G. achilleae), milfoil cyst nematodes (G. millefolii), applecyst nematodes (G. mali), white cyst potato nematodes (G. pallida),golden nematodes (G. rostochiensis), tobacco cyst nematodes (G.tabacum), Osborne's cyst nematodes (G. tabacum solanacearum),horsenettle cyst nematodes (G. tabacum virginiae), pin nematodes(Gracilacus spp., G. idalimus), spiral nematodes (Helicotylenchus spp.,H. africanus, H. digonicus, H. dihystera, H. erythrinae, H.multicinctus, H. paragirus, H. pseudorobustus, H. solani, H.spicaudatus), sheathoid nematodes (Hemicriconemoides spp., H. biformis,H. californianus, H. chitwoodi, H. floridensis, H. wessoni), sheathnematodes (Hemicycliophora spp., H. arenaria, H. biosphaera, H.megalodiscus, H. parvana, H. poranga, H. sheri, H. similis, H.striatula), cyst nematodes (Heterodera spp.), almond cyst nematodes (H.amygdali), oat (or cereal) cyst nematodes (H. avenae), Cajanus (orpigeon pea) cyst nematodes (H. cajani), Bermuda grass (or heart-shaped,or Valentine) cyst nematodes (H. cardiolata), carrot cyst nematodes (H.carotae), cabbage cyst nematodes or brassica root eelworm (H.cruciferae), nutgrass (or sedge) cyst nematodes (H. cyperi), Japanesecyst nematodes (H. elachista), fig (or ficus, or rubber) cyst nematodes(H. fici), galeopsis cyst nematodes (H. galeopsidis), soybean cystnematodes (H. glycines), alfalfa root (or pea cyst) nematodes (H.goettingiana), buckwheat cyst nematodes (H. graduni), barley cystnematodes (H. hordecalis), hop cyst nematodes (H. humuli), Mediterraneancereal (or wheat) cyst nematodes (H. latipons), lespedeza cyst nematodes(H. lespedezae), Kansas cyst nematodes (H. longicolla), cereals rooteelworm or oat cyst nematodes (H. major), grass cyst nematodes (H.mani), lucerne cyst nematodes (H. medicaginis), cyperus (or motha) cystnematodes (Heterodera mothi), rice cyst nematodes (H. oryzae), Amu-Darya(or camel thorn cyst) nematodes (H. oxiana), dock cyst nematodes (H.rosii), rumex cyst nemtodes (H. rumicis), sugar beet cyst nematodes (H.schachtii), willow cyst nematodes (H. salixophila), knawel cystnematodes (H. scleranthii), sowthistle cyst nematodes (H. sonchophila),tadzhik cyst nematodes (H. tadshikistanica), turkmen cyst nematodes (H.turcomanica), clover cyst nematodes (H. trifolii), nettle cyst nematodes(H. urticae), ustinov cyst nematodes (H. ustinovi), cowpea cystnematodes (H. vigni), corn cyst nematodes (H. zeae), rice root nematodes(Hirschmanniella spp., H. belli, H. caudacrena, H. gracilis, H.oryzae),lance nematodes (Hoplolaimus spp.), Columbia nematodes (H. columbus),Cobb's lance nematodes (H. galeatus), crown-headed lance nematodes (H.tylenchiformis), pseudo root-knot nematodes (Hypsoperine graminis),needle nematodes (Longidorus spp., L. africanus, L. sylphus), ringnematodes (Macroposthonia (Mesocriconema xenoplax), cystoid nematodes(Meloidodera spp.), pine cystoid nematodes (M floridensis), tadzhikcystoid nematodes (M tadshikistanica), cystoid body nematodes(Meloidoderita spp.), stunt nematodes (Merlinius spp., M. brevidens, M.conicus, M. grandis, M. microdorus), root-knot nematodes (Meloidogynespp., M. acronea, M arenaria, M.artiellia, M. brevicauda, M. camelliae,M. carolinensis, M. chitwoodi, M. exigua, M. graminicola, M. hapla, M.hispanica, M. incognita, M. incognita acrita, M. indica, M. inornata, M.javanica, M. kikuyuensis, M. konaensis, M. mali, M. microtyla, M. naasi,M ovalis, M platani, M. querciana, M. sasseri, M tadshikistanica, M.thamesi), knapweed nematodes (Mesoanguina picridis), Douglas firnematodes (Nacobbodera chitwoodi), false root-knot nematodes (Nacobbusaberrans, N. batatiformis, N. dorsalis), sour paste nematodes(Panagrellus redivivus), beer nematodes (P. silusiae), needle nematodes(Paralongidorus microlaimus), spiral nematodes (Pararotylenchus spp.),stubby-root nematodes (Paratrichodorus allius, P. minor, P. porosus, P.renifer), pin nematodes (Paratylenchus spp., P. baldaccii, P.bukowinensis, P. curvitatus, P. dianthus, P. elachistus, P. hamatus, P.holdemani, P. italiensis, P. lepidus, P. nanus, P. neoamplycephalus, P.similis), lesion (or meadow) nematodes (Pratylenchus spp., P. alleni, P.brachyurus, P. coffeae, P. convallariae, P. crenatus, P. flakkensis, P.goodeyi, P. hexincisus, P. leiocephalus, P. minyus, P. musicola, P.neglectus, P. penetrans, P. pratensis, P. scribneri, P. thornei, P.vulnus, P. zeae), stem gall nematodes (Pterotylenchus cecidogenus),grass cyst nematodes (Punctodera punctate), stunt nematodes(Quinisulcius acutus, Q. capitatus), burrowing nematodes (Radopholusspp.), banana-root nematodes (R. similis), rice-root nematodes (R.oryzae), red ring (or coconut, or cocopalm) nematodes (Rhadinaphelenchuscocophilus), reniform nematodes (Rotylenchulus spp., R. reniformis, R.parvus), spiral nematodes (Rotylenchus spp., R. buxophilus, R.christiei, R. robustus), Thorne's lance nematodes (R. uniformis),Sarisodera hydrophylla, spiral nematodes (Scutellonema spp., S.blaberum, S. brachyurum, S. bradys, S. clathricaudatum, S. christiei, S.conicephalum), grass root-gall nematodes (Subanguina radicicola), roundcystoid nematodes (Thecavermiculatus andinus), stubby-root nematodes(Trichodorus spp., T. christiei, T. kurumeensis, T. pachydermis, T.primitivus), vinegar eels (or nematodes) (Turbatrix aceti), stunt (orstylet) nematodes (Tylenchorhynchus spp., T. agri, T. annulatus, T.aspericutis, T. claytoni, T.ebriensis, T. elegans, T. golden,T.graciliformis, T. martini, T. mashhoodi, T. microconus, T. nudus, T.oleraceae, T. penniseti, T. punensis), citrus nematodes (Tylenchulussemipenetrans), dagger nematodes (Xiphinema spp., X. americanum, X.bakeri, X. brasiliense, X. brevicolle, X. chambersi, X. coxi, X.diversicaudatum X. index, X. insigne, X. nigeriense, X. radicicola, X.setariae, X. vulgarae, X. vuittenezi). In an even more particularembodiment, the nematodes, include but are not limited to Meloidogyneincognita (root knot nematodes), as well as Globodera rostochiensis andGlobodera pailida (potato cyst nematodes); Heterodera glycines (soybeancyst nematode); Heterodera schachtii (beet cyst nematode); andHeterodera avenae (cereal cyst nematode) and Rotylenchulus reniformis(reniform nematodes) (Pratylenchus spp.) (lesion nematodes),Belonolaimus sp. (sting nematode), Hopolaimus sp. (lance nematode),Bursaphalenchus spp., (e.g., pine wood nematodes), and Ditylenchus spp.(e.g., stem nematodes).

Application of an effective pesticidal controlling amount of aChromobacterium sp., Burkholderia sp., and/or Flavobacterium sp. isprovided. A substantially pure culture, whole cell broth, supernatant,filtrate, extract or compound of the bacterial strain may be applied,alone or in combination with another pesticidal substance, in aneffective pest control or pesticidal amount. An “effective amount” isdefined as the quantity of microorganism cells, supernatant, whole cellbroth, filtrate, cell fraction or extract, metabolite and/or compoundalone or in combination with another pesticidal substance that issufficient to modulate plant parasitic nematode infestation. The amountthat will be within an effective range can be determined by laboratoryor field tests by those skilled in the art. In an embodiment of theinvention, when the composition is applied directly to the seed, theeffective amount is a concentration of about 0.01%-30% of the activeingredient (for example, the Chromobacterium sp., Burkholderia sp., orFlavobacterium sp.) per 100g of seed. In another embodiment, theeffective amount is a concentration of about 0.05%-25%, or about0.1%-20%, or about 0.5%-15%, or about 1%-10%, or about 2%-5% of theactive ingredient per 100 g of seed. In yet another embodiment, theeffective amount is a concentration of about 0.5% to 1% of the activeingredient per 100 g of seed. In another embodiment of the invention,when the composition is applied to the soil by, for example, in furrow,the effective amount is about 0.1-50 oz of the active ingredient per1000 ft row. In other embodiments, the effective amount for soilapplication is about 1-25 oz of the active ingredient per 1000 ft row.In yet another embodiment, the effective amount is about 2-20 oz, orabout 3-15oz, or about 4-10 oz, or about 5-8 oz, of the activeingredient per 1000 ft row.

The compositions, substantially pure culture, whole cell broth,supernatant, filtrate extract or compounds set forth above may modulateplant parasitic nematode infestation by a number of mechanisms. Theseinclude but are not limited to exterminating the plant parasiticnematodes at various stages of the life cycle (eggs, larvae or adult),affecting the motility of the plant parasitic nematodes, and/or reducingthe number and/or viability of eggs that are laid by the parasiticnematodes.

The said formulated product can be used alone or sequentially with oneor more other components as set forth above, such as growth promotingagents in a tank mix or in a program (sequential application calledrotation) with predetermined order and application interval during thegrowing season.

Contact of the plants with the compositions set forth above may becarried out directly or by allowing the compositions to act on theirsurroundings, habitat or storage space by, for example, immersion,spraying, evaporation, fogging, scattering, painting on, injecting orsoil amendment. In the case that the composition is applied to a seed,the composition may be applied to the seed as one or more coats prior toplanting the seed using one or more coats using methods known in theart.

The compositions comprising a Chromobacterium sp., Burkholderia sp.,and/or Flavobacterium sp. may be applied by root dip at transplanting,specifically by treating a fruit or vegetable with the composition bydipping roots of the fruit or vegetable in a suspension of saidcomposition (about 0.25 to about 1.5% and more particularly about 0.5%to about 1.0% volume by volume) prior to transplanting the fruit orvegetable into the soil.

Alternatively, the composition may be applied by drip or otherirrigation system. Specifically, the composition comprising aChromobacterium sp., Burkholderia sp., and/or Flavobacterium sp. may beinjected into a drip irrigation system. In a particular embodiment, thecomposition may be applied at a rate of about 11 to about 4 quarts peracre.

In yet another embodiment, the composition may be added as an in-furrowapplication. Specifically, the composition may be added as an in-furrowspray at planting using nozzles calibrated to deliver a total output of2-6 gallons/acre, or at outputs of up to 20 gallons per acre. Nozzlesare placed in the furrow opener on the planter so that the pesticideapplication and seed drop into the furrow are simultaneous. The mixturesset forth above and, where appropriate, a solid or liquid adjuvant areprepared in known manner. For example, the mixtures may be prepared byhomogeneously mixing and/or grinding the active ingredients withextenders such as solvents, solid carriers and, where appropriate,surface-active compounds (surfactants). The compositions may alsocontain further ingredients such as stabilizers, viscosity regulators,binders, adjuvants as well as fertilizers or other active ingredients inorder to obtain special effects.

EXAMPLE

The composition and methods set forth above will be further illustratedin the following, non-limiting Examples. The examples are illustrativeof various embodiments only and do not limit the claimed inventionregarding the materials, conditions, weight ratios, process parametersand the like recited herein.

A seed treatment trial was conducted to determine the effect of wholecell broths from Flavobacterium sp. H492 (MBI-302), Chromobacteriumsubtsugae sp. nov. (MBI-203), Burkholderia A396 (MBI-206), and enrichedwhole cell broth from Flavobacterium sp. H492 (MBI-302) on Heteroderaglycines (soybean cyst nematode, SCN) infestation of soybean plants.

The study included six experimental seed treatments, an untreatedcontrol, and a standard (positive) control. Treatments of the seedsincluded the following:

1. Untreated control

2. Standard—AVICTA® Complete Soybean (Syngenta) 4.17 FS applied at 0.15mg active ingredient per seed

3. Flavobacterium sp. H492 (MBI-302) @1 gram active ingredient per 100grams seed

4. Flavobacterium sp. H492 (MBI-302) “enriched” @1 gram activeingredient per 100 grams seed

5. Chromobacterium subtsugae sp. nov. (MBI-203) @ 1 gram activeingredient per 100 grams seed

6. Burkholderia A396 (MBI-206) @0.5 gram active ingredient per 100 gramsseed

7. Burkholderia A396 (MBI-206) @1.0 gram active ingredient per 100 gramsseed

8. Chromobacterium subtsugae sp. nov. (MBI-203) in-furrow 5 oz per 1000ft row in a minimum of 20 gallons per acre (GPA).

The term “enriched” (as used in treatment 4 above) refers to a MBI-302whole-cell broth in which most of the water has been extracted to yielda concentrated cell paste.

Seed Treatment

Soybean seeds (94Y82) were prepared by weighing out the desired amountof seeds per treatment. Coating material, for application to seeds,consisted essentially of a sticking component (e.g. methyl cellulose)and whole cell broth from each of Flavobacterium sp. H492 (MBI-302),Burkholderia A396 (MBI-206), and Chromobacterium subtsugae sp. nov(MBI-203), enriched whole-cell broth from Flavobacterium sp. H492(MBI-302) or AVICTA® Complete Soybean (Syngenta) (positive control).Negative control seeds were untreated. The amount of coating materialwas 2% of the weight of the seed. The coating material was applied tothe seeds in a seed coater and the seeds were dried overnight.

Nematodes

Heterodera glycines were reared on Hutchinson soybeans for 60 days in agreenhouse, and inoculated into soil just prior to planting.

Planting and Plot Size

The trial was conducted in outdoor microplots (pots approximately 1 footin diameter) having a volume of 19,000 cm³, arranged in a randomizedcomplete block design with 5 replicate plots for each of the eighttreatments. Nematode-free field soil (Wickham fine sandy loam, 70-16-18S-S-C, 1.0% OM, pH=6.5) was used. One week before planting, lime andfertilizer (OSMOCOAT® 10-10-10 Slow Release) (The Scotts Company) wereadded to the soil in each microplot and incorporated with a hand trowel.Lime was added to raise the pH of the soil to 7, which is optimal forsoybean growth.

Soybean seeds, untreated or treated as described above, were planted inthe pots in linear rows, at a density of ten seeds per foot. Immediatelyprior to planting, 500 cm³ of H. glycines-inoculated soil, containing atleast 100 cysts, was mixed into each row. Plots were watered twice aday.

Data recording

Seeds were planted on May 1, 2013. Fifty-seven days after planting (Jul.30, 2013), plant height (distance from soil surface to plant top) wasdetermined in three plants selected randomly from each plot. From thesemeasurements, an average (mean) height was calculated. Mean plantheights for each of the eight treatments are shown in the third columnof Table 1.

The number of cysts associated with each plant was also determined atfifty-seven days after planting. For measurement of cyst number, fourcore samples of 2.5×12 cm were obtained from each pot. The four sampleswere combined and homogenized, and 150 cm³ of the homogenate wasremoved. Cysts were extracted from the 150 cm³ sample by gravityscreening on an 80 mesh sieve and counted. Results, expressed as meancyst number, are shown in the fourth column of Table 1.

Seed production per plot was measured 127 days after planting (i.e.,Oct. 8, 2013). All plants in a plot were cut down and placed in paperbags for three days for drying. Dried plants from each plot werehand-threshed, the seeds were collected and adjusted to 13% moisturecontent, then weighed. Results, expressed as mean seed weight per plot,are shown in the fifth column of Table 1.

TABLE 1 Treatment Concentration Avg. Height Cyst No. Seed Wt. Untreated— 31.8 (a) 35.9 (ab)  84.2 (b) AVICTA ® 34.3 (a) 13.8 (b) 100.8 (ab)MBI-302 1% 33.9 (a) 44.1 (a)  91.3 (b) MBI-302 enr 1% 33.9 (a) 22.4 (ab)105.1 (ab) MBI-203 1% 35.6 (a) 16.4 (ab) 111.3 (ab) MBI-206 0.5%   34.7(a) 20.0 (ab) 113.5 (ab) MBI-206 1% 33.9 (a) 17.6 (ab)  98.0 (a) MBI-203IF 5 oz/10³ ft. 36.4 (a) 17.0 (ab) 136.8 (a) LSD  3.589  0.259  42.72Std. Dev.  3.336  0.241  39.71 CV  9.72 17.78  37.77 Test substanceslisted in the “Treatment” column included a commercial nematicide(AVICTA ®), whole-cell broths from Flavobacterium sp. H492 (MBI-302),Chromobacterium subtsugae sp. nov. (MBI-203), and Burkholderia A396(MBI-206), and enriched whole cell broth from Flavobacterium sp. H492(MBI-302 enr). Test substances were coated onto seeds (soybean 94Y82),prior to planting, at the concentration (g/100 g seed) shown in the“Conc.” column, except for MBI-203 IF which was applied to soil justprior to planting at a concentration of 5 oz per 1000 ft in a minimum of20 gallons per acre. Heights are expressed in cm; seed weights areexpressed in grams per plot. Mean values followed by the same letter donot significantly differ (p = 0.1, Student-Newman-Kuels test) LSD: Leastsignificant difference at 90% confidence level (p = 0.1) Std. Dev.:Standard deviation CV: Coefficient of Variation

Data Analysis

Least significant difference (Fisher's LSD) values were obtained forplant height, cyst number and seed weight values, as shown in Table 1.Values that differ from the control untreated value by more than the LSDindicate that the difference in values has a 90% probability ofresulting from the treatment. By this criterion, both seed coating with,and in-furrow application of, MBI-203 reverse the growth inhibitioncaused by SCN infestation; and both performed better than the commercialnematicide (Table 1, Column 3).

With respect to cyst number, whole-cell broth from MBI-203 and MBI-206,and enriched whole-cell broth from MBI-302 all reduced the number ofcysts associated with infested plants (Table 1, Column 4).

Finally, in-furrow application of MBI-203 increased the amount of seedproduced by SCN-infested plants (Table 1, Column 5).

These results show that application of MBI-203, MBI-206 and/or MBI-302to seeds or soil is effective in reducing the number of SCN cysts in thesoil, after seeds are planted in SCN-infested soil. They also show thatMBI-203 reduces growth inhibition caused by SCN infestation, andincreases seed yield in SCN-infested soybean plants. They also show thatMBI-302 and MBI-206 treated plots are not different from the commercialnematicide in yields per plot.

This invention may be embodied in other forms or carried out in otherways without departing from the spirit or essential characteristicsthereof. The present disclosure is therefore to be considered as in allaspects illustrate and not restrictive, and all changes which comewithin the meaning and range of equivalency are intended to be embracedtherein.

Various references are cited throughout this specification, each ofwhich is incorporated herein by reference in its entirety.

What is claimed is:
 1. A method for modulating soybean cyst nematodeinfestation of a plant comprising contacting the plant with an effectiveamount of a composition comprising a bacterial strain selected fromFlavobacterium sp. H492 (NRRL Accession No. B-50584), Burkholderia A396(NRRL Accession No. B-50319), and Chromobacterium subtsugae sp. nov.(NRRL Accession No. B-30655) to modulate the soybean cyst nematodeinfestation.
 2. The method of claim 1, wherein the contacting comprisesapplying the composition to a seed of the plant prior to planting. 3.The method of claim 1, wherein the contacting comprises adding thecomposition to a substrate in which the plant is grown.
 4. The method ofclaim 3, wherein the substrate is soil.
 5. The method of claim 4,wherein the composition is added to the soil in furrow.
 6. The method ofclaim 1, wherein the composition is selected from a whole cell broth,cell fraction, filtrate, supernatant, compound, and extract of thebacterial strain.
 7. The method of claim 1, wherein the bacterial strainis Flavobacterium sp. H492.
 8. The method of claim 1, wherein thebacterial strain is Burkholderia A396.
 9. The method of claim 1, whereinthe bacterial strain is Chromobacterium subtsugae sp. nov.
 10. Themethod of claim 1, wherein the method reduces the number of soybean cystnematodes in the soil or plant, or on the plant, compared to a plantthat has not been contacted with the composition.
 11. The method ofclaim 1, wherein the method promotes growth of the plant, compared to aplant that has not been contacted with the composition.
 12. The methodof claim 1, wherein the method increases the yield of seeds produced bythe plant, compared to a plant that has not been contacted with thecomposition.
 13. The method of claim 1, wherein the plant is a soybeanplant.
 14. The method of claim 7, wherein the composition is a wholecell broth of Flavobacterium sp. H492.
 15. The method of claim 8,wherein the composition is a whole cell broth of Burkholderia A396. 16.The method of claim 9, wherein the composition is a whole cell broth ofChromobacterium subtsugae sp. nov.